The mechanism of nerve excitation was studied from a macromolecular viewpoint. The miniature responses of squid giant axons were analyzed using a real-time spectrum-analyzer. These responses were found to be sensitive both to tetrodotoxin and to tetraethylammonium ions. This finding seriously challenges the dual-channel hypothesis and presents strong evidence suggesting the existence of only one kind of ion channel in nerve membrane. Another major advance in understanding the mechanism of nerve excitation was made when we discovered that a squid giant axon could remain excitable when perfused both intracellularly and extracellularly with an identical solution containing only a divalent salt such as the salts of cobalt, manganese, nickel or barium. This finding reduces the number of ions involved in the excitation process sufficiently to allow the physico-chemical theory of excitation to be advanced.